Amylase production and partial characterization of crude enzyme preparations from two rhizobia strains (R-926 and R-991) were evaluated. For both the strains, maximal amylase activities were achieved during the early-to-mid- exponential growth phase; both were active over a pH range from 4.5 to 8.5 and temperature from 30 to 50 ºC. None of the ions studied (K+, Na+, Ca2+, Hg2+, Mg2+, Mn2+, Cu2+ and Zn2+) was required for the catalytic activity of strain R-926; amylase activity of strain R-991 was stimulated in the presence of K+, Hg2+ and Zn2+. The surfactants SDS, Triton X-100 and Tween-80 did not have a pronounced inhibitory effect on enzyme activities; SDS and Tween-80 caused the highest stimulatory effects. Amylase activities from the rhizobia strains were reduced by up to 30% in the presence of EDTA; amylase activity of R-926 was also inhibited by HgCl2, suggesting that Ca2+and cysteine residues could be important for activity of this strain.

Amylases are among the most important enzymes used in modern biotechnology, particularly in the processes involving starch hydrolysis. The extensive application of amylases in the food, starch liquefaction and saccharification, detergent, textile, paper, brewing and distilling industries has paved a way for their large-scale commercial production (Gupta et al., 2003). Although amylases originate from different sources (plants, animals and microorganisms), microbial amylases generally meet industrial demands best, due to their short growth period, productivity and thermostability (Burhan et al., 2003). Today, a large number of microbial amylases are available commercially and they have almost completely replaced chemical hydrolysis of starch in the starch processing industry (Pandey et al., 2000).

Microbial amylases are produced mainly from cultures of Aspergillus, Bacillus, Clostridium, Pseudomonas, Rhizopus and Streptomyces species (Pandey et al., 2000). Because of the industrial importance of amylases, there is an increasing worldwide interest in the screening of new microorganisms producing amylases suitable for new industrial applications (Burhan et al., 2003; Gupta et al., 2003). Rhizobia are gram-negative soil bacteria belonging to the family Rhizobiaceae that are capable of infecting and nodulating the roots of their hosts, leguminous plants. Cellulolytic and pectinolytic (Jimenéz-Zurdo et al., 1996), proteolytic (Glenn and Dilworth, 1981) and ureolytic (Toffanin et al., 2002) enzyme activities have been detected in pure rhizobial culture. Nonetheless, studies on rhizobia amylases are still scarce. Recently, Oliveira et al. (2006) reported the occurrence of extracellular amylases in rhizobia strains in solid and liquid media (Oliveira et al., 2007). The aim of this study was the production and characterization of extracellular amylases from two indigenous Central Amazonian rhizobia strains.

The rhizobia strains used in this study were Rhizobium sp. INPA R-926 and Bradyrhizobium sp. INPA R-991 isolated from soils of the Central Amazonian floodplain. The rhizobial strains were identified by partial sequencing of the 16S rRNA gene (Oliveira et al., 2007). Stock cultures were maintained on extract-mannitol agar (YMA) slants (Vincent, 1970) at 4 ºC. The medium used for enzyme production was composed of (g L-1): 10.0 maltose, 0.4 K2HPO4, 0.1 K2HPO4, 0.2 MgSO4.7 H2O, 0.1 NaCl and 0.4 yeast extract. The pH of the medium was adjusted to 6.8 with KOH and autoclaved at 120 ºC for 20 min. Fifty milliliters of medium were taken in 150 mL Erlenmeyer flasks and inoculated with 1 mL of inoculum culture, grown in YM medium (Vincent, 1970) for 3 (R-926 = 1.4 x 109 CFU) and 5 days (R-991 = 2.0 x 109 CFU), and incubated at 28 ºC with rotary shaking (65 cycles per min). Cultures were harvested and centrifuged (12000 rpm, 10 min) at 24-h intervals for 8 (R-926) and 12 days (R-991) of growth. At these intervals, the cell-free culture supernatant served as the enzyme source to evaluate the reducing sugars, amylase activity, total extracellular protein and final pH of the medium. The turbidity of the cultures was determined by measuring the increase in optical density at 560 nm in a Spectrum UV-Vis Spectrophotometer. Biomass production (g. L-1) was also determined by drying the pellet at 105 ºC overnight until constant weight.

For enzymatic characterization studies, the rhizobia strains were incubated for 48 h in the same experimental conditions mentioned above.

Analytical methods

Amylase activity was determined by monitoring starch hydrolysis and production of reducing sugars from starch. The reaction mixture consisted of 300 mL of previously gelatinized corn starch solution (1%), 350 mL of 0.05 M phosphate buffer (pH 6.5) and 350 mL of crude enzyme preparation. After 30 min of incubation at 37 ºC in a water bath, the reducing sugars (glucose equivalents liberated) were estimated by Nelson's modified Somogyi's method (Nelson, 1944). The colour was read at 511 nm using a Spectrum UV-Vis Spectrophotometer. Glucose was used as the standard. The blank contained 300 mL of corn starch solution and 700 mL of 0.05 M phosphate buffer (pH 6.5). One enzyme unit (U) was defined as the amount of enzyme that released 1 μmol of reducing sugar mL-1 min-1 under the standard assay conditions described above. Total extracellular protein was measured by the biuret method (Gornall et al., 1949), using bovine serum albumin (BSA) in the standard curve.

Effect of pH on activity and stability

The effect of pH on enzyme activity was determined by measuring the activity at 37 ºC using citrate - phosphate (0.05 M, pH 3.5 - 5.5), phosphate (0.05 M, pH 6.5 - 7.5) and Tris-HCl (0.05 M, pH 8.5 - 9.5) buffers for 30 min. Stability of enzyme without substrate was also studied by pre-incubating the crude enzyme at different pH values ranging from 3.5 to 9.5 for 24 h, at room temperature, with the residual activity determined under standard conditions.

Effect of temperature on activity and stability

The effect of temperature on enzyme activity was determined by performing the standard assay procedure for 30 min within a temperature range of 30 - 60 ºC in phosphate buffer (0.05 M, pH 7.5). Thermostability in the absence of substrate was investigated at pH 7.5 after pre-incubation of the crude enzyme at temperatures ranging from 30 to 60 ºC for 2 h in a water bath, and then the samples were removed and the remaining activity assayed under standard conditions.

Effect of cations on enzyme activity

The influence of cations (K+, Na+, Ca2+, Hg2+, Mg2+, Mn2+ and Zn2+) on amylase activity was studied by adding them to the reaction solution to final concentrations of 1 and 5 mM. All metals used were in the chloride and sulphate forms. The activity was assayed under optimum conditions of both pH and temperature as required by the rhizobia amylases. Activity in the absence of any additives (control) was taken as 100%.

Effect of surfactants on enzyme activity

To investigate the effect of surfactants on enzyme activity, the detergents, Triton X-100, Sodium Dodecyl Sulfate (SDS) and Tween-80 were added to the enzyme at final concentrations of 1 and 2%. Amylase activity was then measured in the same way as mentioned earlier.

Effect of inhibitors on enzyme activity

The assay procedure was the same as described above except that the reaction mixture included different inhibitors, such as Ethylene Diamine Tetraacetic Acid (EDTA), HgCl2 and p-Methyl Sulphonyl Fluoride (PMSF), at final concentrations of 5 and 10 mM. Amylase activity was determined as described previously.

Experimental design and statistical analyses

The enzymatic assays were organized using a complete randomized design with three replicates and repeated twice for reproducibility. Tukey's test was used to identify means that differed significantly (p < 0.05). Pearson's correlation coefficients were also considered significant at p < 0.05.

RESULTS AND DISCUSSION

Growth curve and amylase production

Maximum biomass production and optical density (DO560) for both the strains were achieved after the 4th day of cultivation (Figs 1 and 2). Bradyrhizobium strain exhibited maximum total extracellular protein on the 2nd day of cultivation, whereas the Rhizobium strain exhibited its maximum on the 7th day of incubation. For both the strains, maximal amylase activities were achieved during the early-to-middle-exponential growth phase and then decreased gradually, as observed for other bacterial amylases (Castro et al., 1999; Aguilar et al., 2000).

Final pH of the culture medium after 72-h growth of Rhizobium strain increased from 6.8 to 7.4 (Fig. 1). The rise of pH could be due to the utilization of organic acids or production of alkaline compounds, as suggested by Cibis et al. (2004) and Rahman et al. (2005). On the other hand, the final pH of the culture medium of Bradyrhizobium strain decreased to 5.7 during this period (Fig. 2). This could be attributed to the production of acids during the bacterial growth.

Correlations among the parameters evaluated

There was no significant correlation between amylase production and final pH of the medium of the Bradyrhizobium strain. On the other hand, amylase production and final pH of the medium were closely associated for the Rhizobium strain (Table 1). Similar findings have been previously documented by Sunna and Hashwa (1990), Wijbenga et al (1991) and Stamford et al. (2001) for other microbial amylases.

Amylase production from the Rhizobium strain presented significant negative correlations with biomass and optical density (OD560). These negative correlations could be because of the higher amylase production that occurred during the early-to-mid-exponential growth phase, while during the late exponential and early stationary growth phase, rhizobial biomass production was higher. The negative correlation observed between amylase production and optical density (OD560) supported this observation (Table 1). On the other hand, correlation between amylase production and bacterial biomass from Bradyrhizobium strain was significantly positive, confirming several authors who also found this type of linear correlation in Bacillus stearothermophilus (Davis et al., 1980), B. amyloliquefaciens (Castro et al., 1993), Nocardiopsis sp. (Stamford et al., 2001), Bacillus sp. (Cordeiro et al., 2002) and in Rhodothermus marinus (Gomes et al., 2003). There was no significant correlation between amylase production and total extracellular protein for either rhizobia strains (Table 1).

Effect of pH on amylase activity and stability

The pH profiles showed a broad range of activity at 37 ºC for 30 min, exhibiting 70-88% of maximum activities in the pH range 4.5-8.5 (Figs 3 and 4). The pH for the optimum activity of amylase from the Rhizobium strain was 7.5 (Fig. 3). A similar result was reported by Cordeiro et al. (2002) for an amylase produced by Bacillus sp. The optimum pH for amylase activity from the Bradyrhizobium strain was also 7.5 (Fig. 4), but significant levels of activity (above 88%) were still detected between pH 5.5-8.5. The amylases produced by several bacterial sources, including Bacillus sp., have a variety of pH profiles. The maximum activity of most of the enzymes earlier reported has been in the pH range 5.0-12.0 (Kim et al., 1995; Leveque et al., 2000; Hagihara et al., 2001; Gomes et al., 2003; Goyal et al., 2005). In spite of earlier reports on the amylase activity at low pH for some amylases (Buonocore et al., 1976; Ohdan et al., 1999), there are few reports on amylases that have maximum activity at pH lower than 5.0 (Vihinen and Mäntsälä, 1989; Matzke et al., 1997; Jorgensen et al., 1997). In this study, the amylases produced by these rhizobia strains retained up to 78% of their maximum catalytic activities at pH 4.5 (Figs 3 and 4).

The rhizobia amylases were very stable, retaining more than 70% of their original activities in the range pH 4.5-9.5 (Figs 3 and 4). The stability of amylase from the Rhizobium strain increased up to pH 6.5 and then decreased gradually above this pH (Fig. 3). Above pH 5.5, the stability of amylase secreted by the Bradyrhizobium strain decreased significantly (Fig. 4). The amylase enzyme produced by the Rhizobium strain retained up to 94% of its original activity at pH 7.5, while the enzyme from the Bradyrhizobium strain had a loss of 16% (Figs 3 and 4). Similar results have been found in other studies (Cordeiro et al., 2002; Gupta et al., 2003; Sajedi et al., 2005).

Effect of temperature on amylase activity and stability

The amylase from the Rhizobium strain showed good catalytic activity from 30 to 50 ºC, with an optimum temperature of 40 ºC (Fig. 5). Above the optimum temperature, amylase activity decreased significantly. The relative activities at 50 and 60 ºC were 65 and 55%, respectively. Optimal temperatures were between 30 and 50 ºC for amylase from the Bradyrhizobium strain (Fig. 6). At 60 ºC, however, a decrease of 37% in enzyme activity was registered. Several authors have reported that the majority of the bacterial amylases have an optimum temperature in the range of 30-100 ºC, with pH values between 4.6 and 9.0 (Pandey et al., 2000; Cordeiro et al., 2002; Gupta et al., 2003). Based in these studies, it could be inferred that the optimal temperatures found in this study were similar or lower than those described for other bacterial amylases.

The amylase produced by the Rhizobium strain retained 95 and 100% of its original activity at 40 and 50 ºC, respectively (Fig. 5). Above 50 ºC, enzyme activity declined very sharply, possibly because of thermal inactivation. Similarly, amylase from the Bradyrhizobium strain was fully stable at 30 ºC, but lost 26, 29 and 78% of its original activity at 40, 50 and 60 ºC, respectively (Fig. 6). These data were comparable to the results documented by other researchers (Hamilton et al., 1999; Pandey et al., 2000; Cordeiro et al., 2002; Bernhardsdotter et al., 2005).

Effect of metal ions on amylase activity

None of the metal ions evaluated was required for the activity of amylase from the Rhizobium strain (Table 2). The following metal ions had a slight inhibitory effect on enzyme activity: Na+ (5 mM), Ca2+ (5 mM), Cu2+ (1 and 5 mM), Mg2+ (1 and 5 mM) and Mn2+ (1 and 5 mM). On the other hand, stronger inhibitory effects were observed in the presence of K+ (5 mM) and Hg2+ ions (5 mM) after 30 min incubation. Inhibitor effects of 30, 27, 24, 23 and 20% were observed in the presence of 1 mM of K+, Hg2+, Na+, Ca2+ and Zn2+, respectively. In addition, Zn2+ (5 mM) also inhibited enzyme activity by 21%. Metal ions play a key role in protein folding or in catalysis.

None of the surfactants tested had a pronounced inhibitory effect on enzyme activities. Only Triton X-100 showed a moderate inhibitory effect (14%) on amylase activity of Rhizobium strain (Table 3). On the other hand, the addition of SDS (1%) and Tween-80 (2%) was found to stimulate activity by 9 and 21%, respectively. SDS had a pronounced effect on amylase activity (65%) of strain R-991, while Triton X-100 and Tween-80 at a concentration of 1mM increased activity by 6 and 22%, respectively (Table 3).

The stimulatory effect of surfactants on amylase activity is controversial. Arnesen et al. (1998) reported a stimulatory effect of Tween-80 (1.3%) on amylase activity of Thermomyces lanuginosus. In the same study, Triton X-100 had no beneficial effect. The activity of amylase was stimulated by the presence of Tween-80 (1%) in Bacillus sp. (Oberoi et al., 2001). In another study, Triton X-100 (0.1 mM) and Tween-80 (0.05 mM) had weak stimulatory effects on amylase and pullulanase activities in Clostridium thermosulfurogenes (Reddy et al., 1999). Similar results were reported by Srisvastava and Baruah (1986) in B.stearothermophilus. On the other hand, addition of SDS (0.03%) and Tween-80 has pronounced effects on amylase activity in Geobacillus thermoleovorans (Rao and Satyanarayana, 2003). A stimulatory effect of SDS (6%) on amylase activity and stability in Bacillus sp. strain TS-23 was documented by Lo et al. (2001). From the previous reports and the present study, it was evident that the same surfactant could have different effect on the activity of the same enzyme in the same microorganism, or on the activity of different enzymes in the same microorganism, as previously discussed by Reddy et al. (1999).

Effect of inhibitors on amylase activity

When EDTA (5 mM) and HgCl2 (5 mM) were included in the reaction mixture, the enzyme activity of Rhizobium strain was reduced by 30 and 47%, respectively (Table 4). EDTA (5 mM) was also found to reduce by 30% amylase activity of Bradyrhizobium strain (Table 4). In contrast and independently of the concentration tested, HgCl2 increased amylase activity of Bradyrhizobium strain. PMSF, a modifier of serine residues, either had no effect or stimulated activity of both rhizobia strains. Similar findings were reported for amylases from Bacillus strains TS-23 (97%) (Lin et al., 1998), ANT-6 (103%) (Buhan et al., 2003) and L1711 (91%) (Bernhardsdotter et al., 2005). The results of the present study suggested, at least in part, that Ca2+ ions and cysteine could be important for activity of the amylase from strain R-926. The importance of cysteine residues for catalysis was previously described for amylases independently of their origin (Pandey et al., 2000; Lo et al., 2001; Díaz et al., 2003).

From the results, it could concluded that the broad range of pH activity and stability profiles, optimum activity at lower pH and the high tolerance towards surfactants such as SDS, Triton X-100 and Tween-80 could make these rhizobial amylases attractive for both basic research and industrial biotechnological processes.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support from the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM Process 624/04) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Process 474007/2003-0). LAO is a CNPq fellow. They thank Dr. Charles Roland Clement (INPA/CPCA) for reviewing the English text.